Secondary bacterial pneumonias complicating influenza and respiratory viral infections are more severe than primary pneumonias and often fatal, but why this occurs is unclear. Our data demonstrate that neutrophils, which are the most abundant white blood cell and critical for fighting bacterial infections, from influenza-infected animals display impaired ability to ingest and kill bacteria, compared to neutrophils from uninfected and bacteria-infected animals. To date, neutrophils have been largely considered to be a homogenous cell population, where the most important factor is whether an infected host has sufficient numbers of neutrophils to fight infection or not. However, our preliminary data suggest that neutrophils can adopt different subtypes - for example, our analysis of gene expression patterns of neutrophils isolated from mouse lung following influenza or Streptococcus pneumoniae infection reveal that neutrophils from virally infected animals (flu-PMNs) significantly differ from neutrophils isolated from bacterially- (S. pneumoniae) infected animals (Sp-PMNs), suggesting that distinct phenotypes of neutrophils emerge in the context of different types of infection. However, neutrophil specialization is a concept that has been poorly recognized and understood particularly in the context of infection, although studies from the cancer literature strongly support this emerging concept. This application tests the hypothesis that neutrophils adopt distinct phenotypes under conditions of viral (influenza) versus bacterial (S. pneumoniae) pneumonia, which is a mechanism contributing to secondary bacterial infections. In addition, we will test the hypothesis that type I interferons, which are a central immune mediator induced by viral infections, lead to the development of the flu-PMN phenotype. The studies in Aim 1 will examine how viral versus bacterial infections regulate critical neutrophil functions over time, including phagocytosis, bacterial killing, reactive oxygen species generation, neutrophil extracellular trap formation, cytokine production, and degranulation responses. In addition, transcriptome changes in neutrophils isolated from the bone marrow, systemic (spleen), and local (lung) compartments of influenza versus S. pneumoniae-infected animals to determine where different neutrophil subtypes develop, and what molecular pathways are activated that might result in different neutrophil phenotypes that emerge during viral and bacterial infection. Finally, the expression pattern of multiple immune receptors will be performed using a powerful novel technique, mass cytometry, to determine whether the balance between activating and inhibitory immune receptors expressed on neutrophils govern changes in neutrophil activities. Aim 2 will examine the in vivo mechanisms underlying how type I interferons regulate the development of the flu-PMN phenotype, and investigate the mechanisms underlying the observation that flu-infected animals who receive neutrophils from bacterially-infected animals have improved ability to fight subsequent bacterial infection compared to their counterparts who receive neutrophils from virally-infected animals. Inflammatory responses in the lung will be determined by examining cell counts and differentials, cytokine levels of lung homogenates over time, and histology. In vivo regulation of phagocytosis and bactericidal activity by exogenous neutrophil administration will be quantified. The results of these studies will identify neutrophil subtypes on the basis of deep phenotyping investigations, as well as help us understand how the effects of type I interferons on neutrophil phenotypes might increase susceptibility to bacterial pneumonia in subjects with flu infection. These findings will be paradigm-shifting to the field of immunology, which largely considers neutrophils as a fairly homogenous effector cell population with limited functionality. In addition, the results will identify new targets that can form the basis for immune regulating therapies aimed at modulating neutrophil function, instead of simply focusing on whether patients have sufficient numbers of neutrophils.